The invention relates to compositions and methods for the treatment of cancer.
Extracellular signals received at transmembrane receptors are relayed into the cells by the signal transduction pathways (Pelech et al., Science 257:1335 (1992)) which have been implicated in a wide array of physiological processes such as induction of cell proliferation, differentiation or apoptosis (Davis et al., J. Biol. Chem. 268:14553 (1993)). The Mitogen Activated Protein Kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular cues into intracellular responses (Nishida et a., Trends Biochem. Sci. 18:128 (1993); Blumer et al., Trends Biochem. Sci. 19:236 (1994)). Many steps of this cascade are conserved, and homologous for MAP kinases have been discovered in different species.
In mammalian cells, the Extracellular-Signal-Regulated Kinases (ERKs), ERK-1 and ERK-2 are the archetypal and best-studied members of the MAPK family, which all have the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dual specificity kinase (Posada et al., Science 255:212 (1992); Biggs III et al., Proc. Natl. Acad. Sci. USA 89:6295 (1992); Garner et al., Genes Dev. 6:1280 (1992)).
Recent studies have identified an additional subgroup of MAPKs, known as c-Jun NH2-terminal kinases 1 and 2 (JNK-1 and JNK-2), that have different substrate specificities and are regulated by different stimuli (Hibi et al., Genes Dev. 7:2135 (1993)). JNKs are members of the class of stress-activated protein kinases (SPKs). JNKs have been shown to be activated by treatment of cells with UV radiation, pro-inflammatory cytokines and environmental stress (Derijard et al., Cell 1025 (1994)). The activated JNK binds to the amino terminus of the c-Jun protein and increases the protein""s transcriptional activity by phosphorylating it at ser63 and ser73 (Adler et al., Proc. Natl. Acad. Sci. USA 89:5341 (1992); Kwok et al., Nature 370:223 (1994)).
Analysis of the deduced primary sequence of the JNKs indicates that they are distantly related to ERKs (Davis, Trends Biochem. Sci. 19:470 (1994)). Both ERKs and JNKs are phosphorylated on Tyr and Thr in response to external stimuli resulting in their activation (Davis, Trends Biochem. Sci. 19:470 (1994)). The phosphorylation (Thr and Tyr) sites, which play a critical role in their activation are conserved between ERKs and JNKs (Davis, Trends Biochem. Sci. 19:470 (1994)). However, these sites of phosphorylation are located within distinct dual phosphorylation motifs: Thr-Pro-Tyr (JNK) and Thr-Glu-Tyr (ERK). Phosphorylation of MAPKs and JNKs by an external signal often involves the activation of protein tyrosine kinases (PTKS) (Gille et al., Nature 358:414 (1992)), which constitute a large family of proteins encompassing several growth factor receptors and other signal transducing molecules.
Protein tyrosine kinases are enzymes which catalyze a well defined chemical reaction: the phosphorylation of a tyrosine residue (Hunter et al., Annu Rev Biochem 54:897 (1985)). Receptor tyrosine kinases in particular are attractive targets for drug design since blockers for the substrate domain of these kinases is likely to yield an effective and selective antiproliferative agent. The potential use of protein tyrosine kinase blockers as antiproliferative agents was recognized as early as 1981, when quercetin was suggested as a PTK blocker (Graziani et al., Eur. J. Biochem. 135:583-589 (1983)).
The best understood MAPK pathway involves extracellular signal-regulated kinases which constitute the Ras/Raf/MEK/ERK kinase cascade (Boudewijn et al., Trends Biochem. Sci. 20, 18 (1995)). Once this pathway is activated by different stimuli, MAPK phosphorylates a variety of proteins including several transcription factors which translocate into the nucleus and activate gene transcription. Negative regulation of this pathway could arrest the cascade of these events.
What are needed are new anticancer chemotherapeutic agents which target receptor tyrosine kinases and which arrest the Ras/Raf/MEK/ERK kinase cascade. Oncoproteins in general, and signal transducing proteins in particular, are likely to be more selective targets for chemotherapy because they represent a subclass of proteins whose activities are essential for cell proliferation, and because their activities are greatly amplified in proliferative diseases.
It is an object of the invention to provide compounds, compositions and methods for the treatment of cancer and other proliferative diseases. The biologically active compounds are in the form of (Z)-styryl benzylsulfones.
It is a further object of the invention to provide intermediates useful for the preparation of compounds having anticancer activity. The intermediates comprise (Z)-styryl benzylsulfides.
The present invention provides for pharmaceutical compositions comprising a pharmaceutically acceptable carrier and one or more compounds of the formula I 
wherein
R1 is selected from the group consisting of hydrogen, chloro and nitro;
R2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, chloro, bromo, iodo and fluoro; and
R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, nitro, chloro, bromo, iodo and fluoro;
provided at least one of R1 or R2 is hydrogen.
According to one embodiment of such compositions, R2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, chloro, bromo and fluoro; and R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, nitro, chloro, bromo and fluoro. According to another embodiment, at least one of R2, R3 and R4 is iodo.
According to one preferred embodiment of the invention, pharmaceutical compositions of compounds of formula I are provided wherein R1 is hydrogen. More preferably, R1 and R3 are hydrogen, and R2 and R4 are independently selected from the group consisting of chloro, fluoro, iodo and bromo, most preferably selected from chloro, bromo and fluoro.
According to another embodiment of the invention, novel compounds of formula I are provided where R1, R2, R3 and R4 are defined as above, provided:
(a) at least one of R1 or R2 is hydrogen;
(b) R1 and R2 may not both be hydrogen when:
(i) R3 and R4 are both hydrogen,
(ii) R3is chloro and R4 is hydrogen, or
(iii) R4is chloro and R3is hydrogen; and
(c) when R1 is hydrogen and R2 is methyl:
(i) both R3 and R4 may not be hydrogen,
(ii) R3 may not be chloro when R4is hydrogen, and
(iii) R4 may not be chloro when R3 is hydrogen.
According to one embodiment of novel compounds, R2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, chloro, bromo and fluoro; and R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, nitro, chloro, bromo and fluoro. According to another embodiment, at least one of R2, R3and R4is iodo.
Preferably, R1 is hydrogen in the novel compounds of the invention. More preferably, R1 and R3 are hydrogen, and R2 and R4 are independently selected from the group consisting of chloro, fluoro, iodo and bromo, most preferably selected from chloro, bromo and fluoro.
According to another embodiment of the invention, novel (Z)-styryl benzylsulfides are provided which are useful as intermediates in the preparation of the biologically active (Z)-styryl benzylsulfones. The (Z)-styryl benzylsulfides have the formula: 
wherein:
R1 is selected from the group consisting of hydrogen, chloro and nitro;
R2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, chloro, bromo, iodo and fluoro, provided that at least one of R1 or R2 is hydrogen;
R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, nitro, chloro, bromo, iodo and fluoro; provided:
(a) at least one of R1 or R2 is hydrogen;
(b) R1 and R2 may not both be hydrogen when:
(i) R3 and R4 are both hydrogen,
(ii) R3 is chloro and R4 is hydrogen, or
(iii) R4 is chloro and R3 is hydrogen; and
(c) when R1 is hydrogen and R2 is methyl:
(i) both R3 and R4 may not be hydrogen,
(ii) R3 may not be chloro when R4 is hydrogen, and
(iii) R4 may not be chloro when R3 is hydrogen.
According to one embodiment of the aforesaid intermediates, R2 is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, chloro, bromo and fluoro; and R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, nitro, chloro, bromo and fluoro. According to another embodiment, at least one of R2, R3 and R4 is iodo.
Preferably, R1 is hydrogen in the aforementioned intermediates. More preferably, R1 and R3 are hydrogen, and R2 and R4 are independently selected from the group consisting of chloro, fluoro, iodo and bromo, most preferably selected from chloro, bromo and fluoro.
Where R2, R3 and/or R4 is halogen, the halogen is preferably selected from the group consisting of chloro, bromo and fluoro.
By xe2x80x9clower alkylxe2x80x9d is meant straight or branched chain alkyl containing from one to six carbon atoms. The preferred alkyl group is methyl. By xe2x80x9clower alkoxyxe2x80x9d is meant straight or branched chain alkoxy containing from one to six carbon atoms. The preferred alkoxy group is methoxy.
According to another embodiment of the invention, a method of treating an individual for cancer or other proliferative disorder is provided, comprising administering to said individual an effective amount of the aforesaid pharmaceutical composition.
In another embodiment, a method of inhibiting growth of tumor cells in an individual afflicted with cancer is provided, comprising administering to said individual an effective amount of the aforesaid pharmaceutical composition.
In another embodiment, a method of inducing apoptosis-of cancer cells, more preferably tumor cells, in an individual afflicted with cancer is provided, comprising administering to said individual an effective amount of the aforesaid pharmaceutical composition.
According to the present invention, certain (Z)-styryl sulfone derivatives selectively kill various tumor cell types without killing normal cells. Without wishing to be bound by any theory, it is believed that the compounds affect the MAPK signal transduction pathway, thereby affecting tumor cell growth and viability. This cell growth inhibition is associated with regulation of the ERK and JNK types of MAPK.
The compounds of the invention have been shown to inhibit the proliferation of various tumor cells by inducing cell death. The compounds are effective against a broad range of tumor types, including but not limited to the following: breast, prostate, ovarian, lung, brain (i.e, glioma) and renal. The compounds are also effective against leukemic cells. The compounds do not kill normal cells in concentrations at which tumor cells are killed.
Treatment of this broad range of tumor cells with the styryl sulfone compounds of the invention leads to inhibition of cell proliferation and induction of apoptotic cell death. In breast tumors, the effect is observed for estrogen receptor (ER) positive as well as estrogen receptor negative cells.
The compounds are also useful in the treatment of non-cancer proliferative disorders, including but not limited to the following: hemangiomatosis in new born, secondary progressive multiple sclerosis, chronic progressive myelodegenerative disease, neurofibromatosis, ganlioneuromatosis, keloid formation, Pagets Disease of the bone, fibrocystic disease of the breast, Peronies and Duputren""s fibrosis, restenosis and cirrhosis.
Tumor cells treated with the compounds of the invention accumulate in the G2/M phase of the cell cycle. As the cells exit the G2/M phase, they appear to undergo apoptosis. Treatment of normal cells with the styryl sulfones does not result in apoptosis.
Both cells treated with the styryl sulfone compounds of the invention and untreated cells exhibit similar levels of intracellular ERK-2, but the biochemical activity of ERK-2, as judged by its ability to phosphorylate the substrate myelin basic protein (MBP), is considerably diminished in drug-treated cell compared to untreated cells. Without wishing to be bound by any theory, these results suggest that the styryl sulfones of the present invention block the phosphorylating capacity of ERK-2.
The styryl sulfones of the present invention enhance the ability of JNK to phosphorylate c-Jun protein compared to mock-treated cells. Without wishing to be bound by any theory, this result suggests that the styryl sulfones may be acting like pro-inflammatory cytokines or UV light, activating the JNK pathway, which in turn may switch on genes responsible for cell growth inhibition and apoptosis.
The compounds of the present invention were prepared by synthetic methods yielding pure compounds in the (Z)-isomeric configuration. Thus, the nucleophilic addition of the appropriate thiols to substituted phenylacetylene with subsequent oxidation of the resulting sulfide by hydrogen peroxide yields the Z-styryl sulfone. The procedure is generally described by Reddy et al., Sulfur Letters 13:83 (1991), the entire disclosure of which is incorporated herein as a reference.
The compounds are named according to the Cahn-lngold-Prelog system, the IUPAC 1974 Recommendations, Section E: stereochemistry, in Nomenclature of Organic Chemistry, Pergamon, Elmsford, N.Y., 1979 (the xe2x80x9cBlue Bookxe2x80x9d).
In the first step of the synthesis, the sodium salt of benzyl mercaptan or the appropriate substituted benzyl mercaptan is allowed to react with phenylacetylene or the appropriate substituted phenylacetylene forming the pure Z-isomer of the corresponding styryl benzylsulfide in good yield.
In the second step of the synthesis, the (Z)-styryl benzylsulfide intermediate is oxidized to the corresponding sulfone in the pure Z-isomeric form by treatment with hydrogen peroxide.
A. Synthesis of Intermediate Sulfides
To a refluxing methanolic solution of substituted or unsubstituted sodium benzylthiolate prepared from 460 mg (0.02 g atom) of (i) sodium, (ii) substituted or unsubstituted benzyl mercaptan (0.02 mol) and (iii) 80 ml of absolute methanol, is added freshly distilled substituted or unsubstituted phenylacetylene. The mixture is refluxed for 20 hours, cooled and then poured on crushed ice. The crude product is filtered, dried and recrystalized from methanol or aqueous methanol to yield a pure (Z)-styryl benzylsulfide.
B. Synthesis of Sulfone
An ice cold solution of a (Z)-styryl benzylsulfide (3.0 g) in 30 ml of glacial acetic acid is treated with 7.5 ml of 30% hydrogen peroxide. The reaction mixture is refluxed for 1 hour and then poured on crushed ice. The separated solid is filtered, dried, and recrystalized from 2-propanol to yield the pure (Z)-styryl benzylsulfone. The purity of the compounds is ascertained by thin layer chromatography and geometrical configuration is assigned by analysis of infrared and nuclear magnetic resonance spectral data.
The styryl sulfones of the invention may be administered in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier. The active ingredient in such formulations may comprise from 0.1 to 99.99 weight percent. By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is meant any carrier, diluent or excipient which is compatible with the other ingredients of the formulation and to deleterious to the recipient.
The compounds of the invention may be administered to individuals (mammals, including animals and humans) afflicted with breast or prostate cancer. The compounds may be administered by any route, including oral and parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, or subcutaneous administration. The active agent is preferably administered with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice.
The active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Gennaro Alphonso, ed., Remington""s Pharmaceutical Sciences, 18th Ed., (1990) Mack Publishing Co., Easton, Pa. Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.
For parenteral administration, the active agent may be mixed with a suitable carrier or diluent such as water, an oil, saline solution, aqueous dextrose (glucose) and related sugar solutions, or a glycol such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain a water soluble salt of the active agent. Stabilizing agents, antioxidizing agents and preservatives may also be added. Suitable antioxidizing agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
For oral administration, the active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, or other suitable oral dosage forms. For example, the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods.
The specific dose of compound according to the invention to obtain therapeutic benefit will, of course, be determined by the particular circumstances of the individual patient including, the size, weight, age and sex of the patient, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration. For example, a daily dosage of from about 0.05 to about 50 mg/kg/day may be utilized. Higher or lower doses are also contemplated.